The role of intermediate (IK) and small (SK) conductance, Ca2+-activated K+ channels has been unequivocally demonstrated in the endothelial-dependent relaxation of vascular smooth muscle. Both IK and SK channels are activated during agonist-induced vasodilation, and knockout of these channels is associated with increased blood pressure. Also, the expression of these channels in endothelia has been shown to be compromised following balloon angioplasty. Finally, these channels are known to be critical to EDHF-mediated vasodilation, which is compromised in a host of cardiovascular diseases. These results have led to the proposal that the pharmacological activation of endothelial IK and SK channels would be of clinical benefit in a wide array of cardiovascular diseases. To fully appreciate the role of IK and SK channels in endothelial function and how they may be manipulated for clinical benefit requires us to answer the following questions: How are the number of IK1 and SK3 channels maintained at the plasma membrane, is this regulated by vasoactive agonists, what is the route of endocytosis for these channels and do these channels recycle back to the membrane or are they targeted for lysosomal degradation? To date, the answers to these critical physiological questions remain completely unknown and thus represent an important gap in our understanding of how these channels modulate the EDHF response in both health and disease. Based on these gaps in our knowledge, we propose the following aims: (i) Define the mechanism by which IK1 is endocytosed and targeted for lysosomal degradation in endothelial cells and whether this is modulated by vasoactive compounds or sheer stress. (ii) We will define the molecular mechanisms involved in the endocytic recycling of SK3 as well as the role of deubiquitylating enzymes in this process. (iii). We will define the ubiquitin-protein ligases (E3) involved in the ubiquitylation and degradation of IK1 and SK3. We will utilize a combination of molecular, protein biochemical and live-cell fluorescence imaging techniques to carry out these studies. The results of these studies will clearly define the mechanism of IK1 and SK3 endocytosis, whether this is altered by physiologic agonists and whether this can be manipulated pharmacologically. Given that the number of channels at the plasma membrane is deterministic in the physiological response of the cell, determining whether these trafficking events can be manipulated for therapeutic benefit is of clinical import. PUBLIC HEALTH RELEVANCE: Both intermediate (IK) and small (SK) conductance, calcium-activated potassium channels are expressed in microvascular endothelial cells where they play a crucial role in maintaining vascular tone and hence blood pressure (1, 2). Based upon these observations, it is clear that modulating the activity or number of IK and/or SK channels would alter vascular tone and therefore be therapeutically beneficial. Our proposed research will define the mechanisms by which IK1 and SK3 channels are endocytosed, recycled and/or targeted for lysosomal degradation in endothelial cells, thereby altering channel number. Our studies will provide novel insight into how these processes can be manipulated for clinical gain.